Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: (A) Representative fluorescent microscopy images of fixed cells stained with fluorescein-labeled SNA or MAL I. Relative fluorescence level of (B) SNA- and (C) MAL I, stained live cells quantified by flow cytometry. MFI intensities were normalized relative to HEK WT Sia expression (B, C). Background (unstained) subtracted MFI values were analyzed using PRISM software; MFI = Mean fluorescent intensity. *, Error bars show mean ± standard mean error. Statistics were calculated using ANOVA. P<0.05; ****P<0.0001; n=6. (D) Unmodified (WT), and Slc35A1 KO A549 cells transduced with an Slc35A1 encoding lentivirus (LV- Slc35A1 ) were fixed and stained with biotinylated SNA or MAL II (magenta) and DAPI (blue). The Alexa Fluor 647-Streptavidin signal intensities were assessed via microscopy. Images are representative of n=3 independent replicates. Scale bar represents 50µm. (E, F) Cells from (D) were incubated with biotinylated SNA or MAL II and the APC-streptavidin in live cells analyzed via flow cytometry. Following subtraction of the median APC signal of corresponding background samples, the median APC signal was normalized to that of WT cells. Data are means +/- standard error of mean/standard deviation from n=3 independent experiments. Statistical significance was inferred by two-tailed one sample t test with a theoretical mean of 100. ***P<0.001, **P< 0.01.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Microscopy, Staining, Labeling, Fluorescence, Flow Cytometry, Expressing, Software, Transduction, Incubation, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: (A) Representative fluorescent microscopic images of fixed cells incubated with HA-hFc probe and detected with fluorescein-labelled anti-human Fc. (B) Relative fluorescent intensities of live HA-hFc-stained cells quantified by flow cytometry. Fluorescent intensities were normalized relative to fTfR-5J8 expressing cells. (C) The fTfR and fTfR-5J8 expression was analyzed in A549 Slc35A1 KO cells transduced with lentiviruses encoding fTfR (LV-fTfR) or fTfR-5J8 (LV-fTfR-5J8) via qPCR. Primers targeting GAPDH, the extracellular domain of fTfR and 5J8 were utilized. Data are 40-Ct values from technical triplicates from n=1 experiment. A Ct of 40 was assigned to samples where no amplification was detected. (D) Cells from (C) and A549 Slc35A1 KO cells stably expressing SLC35A1 (LV- Slc35A1 ) were inoculated with A/Netherlands/602/09 at a MOI 25 for 1.5hr on ice. Cells were incubated with an anti-HA antibody and the signal from bound virus quantified via flow cytometry. A representative histogram from n=3 independent experiments is shown. (E) Quantification of the percentage of IAV positive cells from (D). The gating strategy was established using the mock-infected sample. Data are means ± standard deviation and statistical significance was inferred by one-way ANOVA with Sidak’s multiple comparisons test. ****P<0.0001.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Incubation, Staining, Flow Cytometry, Expressing, Transduction, Amplification, Stable Transfection, Virus, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: (A) Representative fluorescence microscopy images of fixed Ca’09 infected cells. Receptors (fTfR-5J8 and full-length fTfR) were transiently expressed on HEK Slc35A1 KO for 24hrs before viral infection (MOI = 0.2) for 8hrs. Cells were fixed and stained with anti-NP antibody (green) and DAPI (blue). (B and C) Infection data for Ca’09 or PR8 infected cells respectively. Four different fields of view were imaged for each condition and % infection was determined. Data was normalized to HEK WT infected cells. (D) Infection curve for the Neth/09 -Renilla at MOI = 3 in A549 LV-fTfR, LV-fTfR-5J8 and LV- Slc35A1 cells. (E) AUC plot from D, where AUC values of LV-fTfR-5J8 and LV-fTfR are shown relative to LV- Slc35A1. ND = No infection detected, NS = not significant, AUC = Area under the curve, MOI = Multiplicity of infection. All experiments were performed in three independent replicates (n=3). Error bars show mean ± standard mean error. Statistics were calculated using ANOVA. *P<0.05; **P<0.01, ****P<0.0001.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Fluorescence, Microscopy, Infection, Staining

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: A549 Slc35A1 KO cells stably expressing SLC35A1 (LV- Slc35A1 ) or fTfR-5J8(LV-5J8-TfR) were pre-treated with Dynasore (40-20µM), Pitstop 2 (15-10µM), NH 4 Cl (25mM) or DMSO (0.15%) for 30 minutes at 37°C and then infected with Neth/09- Renilla at a MOI 3 in their presence. (A) Cell viability at 24 hours post-inhibitor treatment relative to the DMSO control. (B) Neth/09 -Renilla infection curves. Dashed line indicates luminescence from the mock-infected sample. (C) AUC values from (B) were normalized to the DMSO-treated sample within each cell line. (A-C) Data are means ± standard deviation from n=3 independent experiments. Statistical significance in (C) was determined by two-tailed one sample t-test with a theoretical mean of 100. *P<0.05, ns = not significant.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Stable Transfection, Expressing, Infection, Control, Standard Deviation, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Single continuous lumen formation in the zebrafish gut is mediated by smoothened -dependent tissue remodeling

doi: 10.1242/dev.100313

Figure Lengend Snippet: Generation of an intestine-specific transgenic line. (A,B) Dorsal, top panel, and lateral view, bottom panel, in situ hybridization showing claudin 15-like a expressed specifically in the intestine at 56 hpf. (C) Schematic of TgBAC(cldn15la-GFP) generation. The recombination target is shown on top, and the expected protein structure is shown on the bottom. (D) Confocal cross-section of a 72 hpf TgBAC(cldn15la-GFP) embryo. (E) Magnification of box from D. (F) Immunolocalization of Cldn15la to the basolateral membranes of intestinal epithelial cells. (G,H) Whole-mount fluorescent images of 55 hpf and 75 hpf embryos expressing TgBAC(cldn15la-GFP). Arrows indicate the gut tube. (I-L) Live imaging of TgBAC(cldn15la-GFP) using SPIM. Snapshots from a single plane from 48-70 hpf. Arrowheads indicate lumens; arrows point to cell-cell contacts between lumens. (M) Stitched confocal whole-mount images of a TgBAC(cldn15la-GFP) embryo show unfused lumens (arrowhead) in the posterior intestine at 68 hpf that are separated by cell-cell contacts (arrow). (N) Magnification of cell-cell contacts from M. Arrows indicate contacts. Phalloidin (red). Scale bars: 50 μm in D-F; 20 μm in M,N.

Article Snippet: The cldn15la-GFP BAC was linearized using AsiSI (NEB), injected into one-cell stage embryos to generate TgBAC ( cldn15la-GFP ) pd1034 .

Techniques: Transgenic Assay, In Situ Hybridization, Expressing, Imaging

Journal: Development (Cambridge, England)

Article Title: Single continuous lumen formation in the zebrafish gut is mediated by smoothened -dependent tissue remodeling

doi: 10.1242/dev.100313

Figure Lengend Snippet: Basolateral adhesions separate lumens along the AP axis. (A-A′′) Whole-mount confocal image of an embryo expressing GFP-Podocalyxin (false colored in red) and stained for cadherin (green) shows luminal expansion during a fusion event. Ras-RFP (white) marks cell membranes. Arrows mark fusion event. Blue, DAPI. (B-B′′) Optical sections from a z-stack surrounding a fusion event. (C-C′) Whole-mount confocal image of a TgBAC(GFP-cldn15la) embryo shows a putative adhesion snapping event during fusion. The arrow marks adhesion at the surface. (D-D′) Whole-mount confocal image of an embryo expressing GFP-Podocalyxin (red) and stained for cadherin (green) shows adhesion snapping during fusion. Ras-RFP (white) marks cell membranes. The arrow marks adhesion at the surface. (E-E′) Optical sections from z-stack surrounding a fusion event. The asterisk marks a cell with adhesion. (F,F′) Space-fill projection labeling cells surrounding the fusion event. Lumen, red. The asterisk marks a cell with adhesion. Scale bars: 10 μm.

Article Snippet: The cldn15la-GFP BAC was linearized using AsiSI (NEB), injected into one-cell stage embryos to generate TgBAC ( cldn15la-GFP ) pd1034 .

Techniques: Expressing, Staining, Labeling

Journal: Development (Cambridge, England)

Article Title: Single continuous lumen formation in the zebrafish gut is mediated by smoothened -dependent tissue remodeling

doi: 10.1242/dev.100313

Figure Lengend Snippet: smos294 mutants exhibit a lumen fusion defect. (A-H) Confocal cross sections of wild-type (top) and smos294 (bottom) intestines at 72 hpf, 85 hpf, 96 hpf and 110 hpf. Phalloidin (red). (I,J) Confocal whole-mount image of wild-type and smos294 embryos expressing TgBAC(cldn15la-GFP) to highlight the cellular and luminal architecture of the intestine at 72 hpf. (I) Wild-type intestine, (J) smos294 intestine. Arrowheads, lumens; asterisk, adjacent lumens. Scale bars: 20 μm.

Article Snippet: The cldn15la-GFP BAC was linearized using AsiSI (NEB), injected into one-cell stage embryos to generate TgBAC ( cldn15la-GFP ) pd1034 .

Techniques: Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Specific recognition of hyaluronan by the Ig-like-V-set domain of hSiglec-9. (a) Immobilized high molecular weight-hyaluronan (HMW-HA) was probed with human Siglec–Fc and CD44–Fc chimeras and binding evaluated by using an anti-human IgG-HRP. (b) Binding of hSiglec-9–Fc to immobilized HMW-HA was performed in the presence of increasing concentrations of HMW-HA, heparan sulfate, chondroitin sulfate or heparin. (c, d) To map the hSiglec-9 domain responsible for HA recognition, binding of hSiglec-9–Fc to immobilized HMW-HA was compared to binding of hSiglec-9–Fc with an Arg→Ala mutation in the V-set domain (hSiglec-9R120K), a fusion protein construct of the hSiglec-9 V-set domain + the second Ig-like domain (C2-set) of hSiglec-7 + human IgG Fc tail (hSiglec-9V-7C2–Fc), a fusion protein construct encompassing V-set domain of hSiglec-7 + C2-set of hSiglec-9 + human IgG Fc tail (Siglec-7V-9C2–Fc), a fusion protein construct of the hSiglec-9 V-set domain + first C2-set domain + human IgG Fc tail (Siglec-9–Fc 2D). Results are expressed as the mean ± SD. All experiments were performed in triplicate, repeated 3 times (a, d) or 2 times (b). One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Mutagenesis, Construct, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: High molecular weight hyaluronan (HMW-HA) is recognized by hSiglec-9 on human neutrophils. (a) To evaluate the molecular size of hyaluronan (HA) responsible for binding to hSiglec-9, high molecular weigh HA (HMW-HA; >1,000 kDa), low molecular weigh (200 – 300 kDa-HA and 25 – 75 kDa-HA), nano-HA (nonamers) and oligo-HA (hexamers) fragments were added to compete with binding of hSiglec-9–Fc to immobilized HMW-HA plates. Binding was evaluated using an anti-human IgG-HRP. Experiment performed in triplicate and repeated 3 times; results are expressed as mean ± SD. (b) Flow cytometry reveals constitutive expression of Siglec-9 and CD44 on human neutrophils from 9 different donors; geometric mean ± 95% confidence interval. (c) Binding of FITC-labeled HMW-HA to neutrophils of these donors was evaluated by flow cytometry; geometric mean ± 95% confidence interval. (d) Human neutrophils were pretreated with anti-Siglec-9 monoclonal antibodies, anti-human CDw329 (BD Pharmingen, #550906) and anti-human Siglec-9 (R&D Systems, #BAF1139) and effects on binding to FITC-labeled HMW-HA determined. CDw329 Ab blocked binding of HMW-HA, but not GBS capsule (Fig. 5b) and was designated “α-Sig-9(HA)”; in contrast, the R&D Systems Ab blocked recognition of GBS capsule (Fig. 5b), but did not interfere with binding to HMW-HA, and was thus designated “α-Sig-9(Sia)”. Data pooled from five independent experiments in triplicate; data represent the mean ± SD. (e, f) Calcein-labeled human neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) and α-CD44 mAbs, added to wells coated with immobilized HMW-HA to facilitate adherence, then unbound neutrophils washed away. Remaining neutrophils were lifted and enumerated. Experiment performed in triplicate and repeated 5 times; results are expressed as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Flow Cytometry, Expressing, Labeling, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) engages hSiglec-9 via its surface hyaluronan capsule. (a) Human Siglec-9–Fc was immobilized to ELISA wells using protein A, and binding of FITC-labeled forms of WT GAS, its isogenic HA capsule-deficient mutant (ΔhasA), an animal-passaged hyperencapsulated (AP) derivative and its isogenic HA capsule-deficient mutant (AP ΔhasA) were evaluated; results are expressed as mean ± SD and repeated 5 times in triplicate with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) (b) Human Siglec-9–Fc chimera was immobilized to ELISA wells via protein A in the presence of α-Sig-9(HA), α-Sig-9(Sia) or isotype control Abs and binding of FITC-labeled WT GAS or sialic acid-expressing serotype III group B Streptococcus (GBS) evaluated. (c) Human Siglec-9/9R120K/5/6/7/11 and human CD44-Fc chimera were immobilized to ELISA wells via protein A. Binding of FITC-labeled WT GAS was evaluated. Results represent mean ± SD; triplicate wells, representative experiment depicted of 5 independent repeats with similar results, performed in triplicate. (d) hSiglec-9–Fc was immobilized to ELISA plates using protein A, then wells were pretreated with HMW-HA, HMW-heparan sulfate or heparin over the indicated range of concentrations. Binding of FITC-labeled WT GAS was evaluated.

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Mutagenesis, Comparison, Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: HMW-HA binding to hSiglec-9 induces SHP-1 recruitment and blunts neutrophil NET production and oxidative burst. (a) 1.2 × 107 human neutrophils were incubated for 30 min + 10 µg/ml of high molecular weight hyaluronan (HMW-HA) + 25 nM PMA. Cell lysates were immunoprecipitated with α-Siglec9 and SHP-1 recruitment was visualized by western blot analysis; results were repeated 2 times with similar results; representative experiment with relative densitometry values is shown. (b) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 Abs, incubated with 10 µg/ml of HMW-HA and activated for 30 min with PMA. Reactive oxygen species (ROS) release was measured with the OxyBURST Green H2HFF BSA probe and results expressed as mean fluorescence intensity (MFI) ± SD; experiment repeated 5 times with similar results; representative experiment is shown. (c) Neutrophils were pretreated α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs, then incubated with 10 µg/ml of HMW-HA and activated with PMA for 3 h; production of neutrophil extracellular traps (NETs) visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); representative fields at 20x magnification is shown; experiment performed in triplicate and repeated 5 times. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD; experiment repeated 3 times in triplicate with similar results; representative experiment shown. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Incubation, Molecular Weight, Immunoprecipitation, Western Blot, Fluorescence, Staining, Picogreen Assay, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) binding to hSiglec-9 via its surface HMW-HA capsule blunts neutrophil oxidative burst, NET responses and bactericidal activity. (a, b) Neutrophils were labeled with OxyBURST Green H2HFF BSA in the presence of α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at MOI = 20 for 30 min and oxidative burst measured by FACS; results are expressed as MFI ±SD and repeated twice with similar results; representative experiment is shown. (c) PMA-stimulated neutrophils (5 × 105 cells) were pretreated with α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs and exposed for 3 h to MOI = 10 of GAS (DNase mutant) that had been pretreated or not with hyaluronidase to remove HA capsule and NET production visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); results are repeated 5 times in triplicate, representative fields at 32 × magnification is shown. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD and repeated 2 times with similar results; representative experiment shown. (e) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at multiplicity of infection (MOI) = 10 for 30 min, then cells lysed and dilutions plated on agar for enumeration of colony forming units to evaluate neutrophil killing of GAS. Data represent the mean + SD of triplicates; repeated 4 times with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) or P < 0.05 (*).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Activity Assay, Labeling, Infection, Mutagenesis, Staining, Picogreen Assay, Comparison

Journal: Nature Communications

Article Title: Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions

doi: 10.1038/s41467-022-29855-8

Figure Lengend Snippet: a – d Upon blue light irradiation, Vasa-mCherry-LOV was recruited to actin, resulting in Vasa-less micromeres at the 16-cell stage in the experimental group. A level of translation was detected by FIAsH-EDT 2 at the 16-cell stage and was increased in the micromere-side of the spindle in the control group ( a and b ) whereas decreased in that of the experimental group ( c and d ). A whole Z-section of the cell of interest (0.44 μm per slice; ~90 slices in total) was imaged and analyzed every 1-min and presented as a maximum 2D-projection. A bright green spot (*) is a non-specific debris that tends to fluoresce under the microscope. Each experiment was repeated at least three times and the measurement results of two other embryos are shown in Supplementary Fig. . e , f Localization of β-catenin-GFP was tracked by time-lapse imaging of every 15 min for ~5 h after activation (approximately, ~12 hpf). In controls, GFP-β-catenin enriched into the vegetal most cells ( e , arrows), whereas its localization became uniform or random in the experimental groups ( f , arrows). Arrowheads indicate ectopic localization of GFP-β-catenin. A whole Z-section of the embryo of interest (1 μm per slice; ~50 slices in total) was imaged and is presented as a maximum 2D-projection. The embryos were lightly squashed to completely immobilize during imaging. Therefore, the embryos that successfully formed micromeres under this condition were selected for the analysis and the embryos in a similar angle were compared between control and experimental groups in this study. These experiments were performed at least three independent times. All Scale bars = 10 μm. Source data are provided as a Source Data file.

Article Snippet: Similarly, Dendra2 (Addgene #54694) was subcloned into pSP64-vector and GFP-Spβ-catenin (Logan and McClay, 1999; SPU_009155) was synthesized by IDT (Illinois, USA) and inserted into the same vector as described above. pCS2-2xmCherry-EMTB and pCS2-EMTB-3xGFP were obtained at Addgene (#26741). pSP64-SpNanos-GFP was previously made .

Techniques: Irradiation, Microscopy, Imaging, Activation Assay

Journal: Nature Communications

Article Title: Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions

doi: 10.1038/s41467-022-29855-8

Figure Lengend Snippet: A list of all materials used in this study.

Article Snippet: Similarly, Dendra2 (Addgene #54694) was subcloned into pSP64-vector and GFP-Spβ-catenin (Logan and McClay, 1999; SPU_009155) was synthesized by IDT (Illinois, USA) and inserted into the same vector as described above. pCS2-2xmCherry-EMTB and pCS2-EMTB-3xGFP were obtained at Addgene (#26741). pSP64-SpNanos-GFP was previously made .

Techniques: Recombinant, Labeling, Clone Assay, Plasmid Preparation, Sequencing, Software, Imaging

Journal: Molecular Oncology

Article Title: Selective induction of cancer cell death by VDAC 1‐based peptides and their potential use in cancer therapy

doi: 10.1002/1878-0261.12313

Figure Lengend Snippet: VDAC 1‐based peptides act by interacting with and detaching HK , decreasing ATP levels, and releasing intracellular Ca 2+ . (A–C) The Antp‐ LP 4, Tf‐D‐ LP 4, and R‐Tf‐D‐ LP 4 peptides interact with HK ‐I and HK ‐ II . Purified HK ‐I and HK ‐ II were fluorescently labeled using the NanoTemper BLUE protein‐labeling kit. HK ‐ II (A) or HK ‐I (B) (100 and 830 n m , respectively) was incubated with the Antp‐ LP 4 (●), Tf‐D‐ LP 4 (○), or R‐Tf‐D‐ LP 4 (▲) peptides (1–100 μ m for 20 min). Then, 3–5 μL of the samples was loaded into MST ‐grade glass capillaries, and thermophoresis was measured using a Monolith‐ NT 115 apparatus, as described in . (C) Kd values of HK ‐I and HK ‐ II for the different peptides. (D,E) The Tf‐D‐ LP 4, R‐Tf‐D‐ LP 4, and D‐∆(1‐14)N‐Ter‐Antp peptides induce HK detachment. (D) HeLa cells were transfected with plasmid pEGFP ‐ HK ‐I and after 24 h, incubated with the indicated concentrations of the Tf‐D‐ LP 4 or D‐∆(1‐14)N‐Ter‐Antp peptides for 3 h in serum‐free medium. The final DMSO concentration in control and peptide‐treated cells was 0.07%. Fixed cells were visualized by confocal microscopy (Olympus 1X81). Arrows indicate cells with diffusion of HK ‐I‐ GFP . (E) A549 or HeLa cells were incubated with or without the indicated concentrations of the Tf‐D‐ LP 4 or R‐Tf‐D‐ LP 4 peptides, respectively, for 3 h, treated with digitonin (0.025%) and HK in the cytosolic fraction was analyzed by immunoblotting as described in . Anti‐ GAPDH and anti‐ VDAC 1 antibodies were used to verify the cytosolic and mitochondria extracts, respectively. The levels of HK ‐I in the cytosolic fraction were quantified and are presented as relative units. (F) The R‐Tf‐D‐ LP 4 (black bars) and Tf‐D‐ LP 4 (white bars) peptides reduce cellular ATP levels assayed as described in . Cellular ATP levels are presented as percentage of control. Results = mean ± SE ( n = 3) ** P ≤ 0.01. (G–I) The R‐Tf‐D‐ LP 4 peptide induces release of intracellular Ca 2+ . A549 cells were incubated with 3 μ m (white bars), 5 μ m (gray bars), or 10 μ m (black bars) of the R‐Tf‐D‐ LP 4 peptide for 1, 2.5, or 5 h and harvested, and intracellular Ca 2+ levels ([Ca 2+ ]i) (G) and cell death (H) were assessed. [Ca 2+ ]i levels and cell death were determined using Fluo‐4 and PI , respectively, and flow cytometry. (I) Correlation between cell death induced by 3 μ m of the R‐Tf‐D‐ LP 4 (○) or 5 μ m of the R‐Tf‐D‐ LP 4 (●) peptides and the decrease in [Ca 2+ ]i levels induced by a 2.5‐h incubation with R‐Tf‐D‐ LP 4 (▲) or a 5‐h incubation with R‐Tf‐D‐ LP 4 (▵).

Article Snippet: Purified HK‐I and HK‐II were fluorescently labeled using a NanoTemper BLUE protein‐labeling kit.

Techniques: Purification, Labeling, Incubation, Transfection, Plasmid Preparation, Concentration Assay, Confocal Microscopy, Diffusion-based Assay, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: Crystal structure of the C-terminal part of D. rerio LIN41. a The crystal structure of the DrLIN41 filamin-NHL domains is displayed in a cartoon mode, with a transparent grey surface in two orientations rotated by 90°. The molecule is colored from blue (N terminus) to red (C terminus) to indicate the topology. Protein domains, termini, and β-propeller blades are labeled for better clarity. b Top view of the RNA-binding site of the DrLIN41 NHL domain with the electrostatic surface potential mapped onto the molecular surface. Surface potential is computed by using the APBS plugin implemented in PyMOL ( www.pymol.org ) and is displayed from – 5.0 kT/e (red, acidic) to + 5.0 kT/e (blue, basic). c DrLIN41 filamin-NHL domains in complex with the lin-29A stem-loop RNA. The filamin and NHL domains are shown in cartoon mode in blue, with a white transparent surface. The lin-29A RNA fragment, forming a hairpin, is displayed as a cartoon with nucleotides in different colors (guanine: green, adenine: blue, cytosine: orange, uracil: cyan). d Magnified view of the lin-29A RNA stem loop bound to the DrLIN41 NHL surface (colors as in c ). A diagram detailing the nucleotide composition of the stem loop is shown above

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: Labeling, RNA Binding Assay

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: Molecular interactions underlying LIN41 binding to RNA SLs. a , b Detailed views of interactions between the lin-29A RNA and the DrLIN41 NHL propeller. Nucleotides and protein side chains are highlighted and their directly interacting residues are shown as sticks; the remaining parts are shown as lines (RNA) or ribbons (protein). Hydrogen bonds are presented as dotted lines and hydrophobic interactions as solid lines. Nucleotides are colored as in Fig. , whereas protein side chains are colored according to the mutational analysis. c Schematic representation of the lin-29A RNA hairpin and of its interactions with DrLIN41 NHL residues (type of interaction and color code as in a , b ). d Expression of mutant HsLIN41 proteins did not severely down-regulate Renilla luciferase (RL) reporter expression unlike the wild-type HsLIN41, when a fragment corresponding to the mab-10 condensed 3′-UTR was transplanted into an unregulated 3′-UTR of the reporter construct. Bars in the graph represent the mean between three biological replicates

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: Binding Assay, Expressing, Mutagenesis, Luciferase, Construct

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: The LIN41 response element. a Schematics depicting RNA features used to build the LIN41 Response Element (LRE) model. Considered were all possible bases in the three loop positions (I, II, and III) and all possible base pairs at the stem position 1 (– 1/ + 1). The pairing probability of stem position 1 was determined by the relative occurrence of all possible structures that a particular RNA sequence can acquire. The pairing probabilities were grouped into seven bins on a log2 scale. Combining the sequence and structure features resulted in 2688 (6 × 64 × 7) RNA motif variants. b A heat map showing the average CeLIN41-binding scores from the RNAcompete experiment for all RNAs containing any particular motif variant as described in a . Pairing probability and base pairs at stem position 1 are shown on the left and the right of the heat map respectively. The loop (I, II, and III) sequences are shown in two rows, for clarity, at the bottom of the heat map. The data were clustered based on the CeLIN41-binding score. The overall distribution of pairing probabilities is shown on top of the pairing probability scale. Bottom right: the drawing represents a stem-loop motif, based on the model, referred to as the LIN41 Response Element (LRE). Yellow: data not available (RNA motif variants supported by < 20 oligo sequences)

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: Sequencing, Binding Assay, Variant Assay

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: LIN41 binds to LREs both in vitro and in vivo. a Fluorescence polarization (FP) assays determining binding constants of CeLIN41 to LRE variants in the position III of the loop. Raw FP data of CeLIN41, interacting with a wild-type LRE (SL I in Supplementary Fig. ), a control stem-loop RNA with five nucleotides in the loop, and LREs mutated at loop position III, are shown in units of millipolarization (mP). The equilibrium dissociation constant ( K D ) is shown for the WT LRE. Each data point is a mean of three experiments and the error bars represent the standard deviation. b The FP assays determining binding constants of CeLIN41 to LRE variants in the position 1 of the stem. Raw FP data of CeLIN41, interacting with WT LRE and LREs mutated at stem position 1, are shown in units of millipolarization (mP). Each data point is a mean of three experiments and the error bars represent the standard deviation. The WT LRE data is the same as in a . It is replotted for easy comparison with the mutants. c Contribution of LREs of varying strengths, predicted by the model in Fig. , present in 5′-UTRs, coding sequences (CDS) and 3′-UTRs, to CeLIN41 binding as determined by linear regression. RNA binding was assayed by co-precipitation with CeLIN41, followed by RNA sequencing (RIP-seq). The error bars represent SEs for the coefficients obtained from the linear regression

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: In Vitro, In Vivo, Fluorescence, Binding Assay, Control, Standard Deviation, Comparison, RNA Binding Assay, RNA Sequencing

Journal: Nature Communications

Article Title: Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition

doi: 10.1038/s41467-018-03920-7

Figure Lengend Snippet: RNA binding preferences of LIN41 and Brat. a Left: crystal structure of the DmBrat NHL domain in a complex with a single-stranded linear RNA (PDB 4ZLR ). The protein surface is colored by the electrostatic surface potential from – 8 kT/e (red, acidic) to + 8 kT/e (blue, basic) and the RNA is shown as a cartoon. The approximate footprint of the RNA interaction on the protein surface is shown as a dotted red line. Right: magnified view of the RNA-binding site. A fragment of the interacting protein surface is shown and colored as on the left. The RNA is shown in surface mode, with carbon atoms in green and other atoms in standard colors. Nucleotide positions are labeled as in the RNA sequence displayed below. b Left: crystal structure of DrLIN41 in complex with the lin-29A RNA stem loop. The protein surface is colored as in a . Right: magnified view of the RNA-binding site in an orientation rotated by 90°. The RNA stem loop is shown in surface mode, with carbon atoms in gold and other atoms in standard colors. The corresponding RNA sequence is on the right

Article Snippet: The DNA sequence encoding D. rerio LIN41 filamin and NHL domains was taken from the NCBI reference sequence NM_001301331 (nucleotides 2040–3209) and synthesized (GeneArt) without any codon optimization.

Techniques: RNA Binding Assay, Labeling, Sequencing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

doi: 10.3389/fcimb.2018.00015

Figure Lengend Snippet: TJ disruption is tightly associated with lipid raft-mediated C. jejuni invasion in polarized epithelial cells. (A–F) Caco-2 cells cultured for 7 days on transwells and treated with 4 mM EGTA for 20 min. Following EGTA treatment, cells were incubated in normal culture medium. During the incubation, the TER value (A) and ZO-1 localization (B–F) were evaluated every hour for 3 h. TER values were calculated as the percentage of the TER value for untreated cells. Scale bar = 10 μm. (G) Caco-2 cells treated with 4 mM EGTA for 20 min were incubated in normal culture medium for the indicated time. After incubation, cells were pretreated with 10 mM MβCD for 1 h and infected with C. jejuni for 6 h. The number of intracellular bacteria was measured using a gentamycin protection assay. Results are shown as the mean ± SD; n = 4. Significant difference from the post Ca 2+ reintroduction 0 h group are shown: N.S.; not significant; * P < 0.05; ** P < 0.01.

Article Snippet: Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Techniques: Cell Culture, Incubation, Infection

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

doi: 10.3389/fcimb.2018.00015

Figure Lengend Snippet: C. jejuni invasion is increased by active inflammation in polarized epithelial cells. (A–C) Caco-2 cells were treated with 20 ng/ml TNF-α for 48 h. Following TNF-α treatment, TER values (A) and ZO-1 localization (B,C) were evaluated. Scale bar = 10 μm. (D) Caco-2 cells were treated with 20 ng/ml TNF-α followed by treatment with 10 mM MβCD for 1 h and were infected with C. jejuni for 6 h. The number of intracellular bacteria was measured by gentamycin protection assay. Results are shown as the mean ± SD n = 4. Significant difference from the control or EGTA treated group are shown: N.S.; not significant; ** P < 0.01.

Article Snippet: Antibodies to the following were diluted in 3% BSA and used for Immunofluorescence staining ZO-1 (1:200; BD Bioscience), Alexa Fluor 568 (1:200, Molecular Probes).

Techniques: Infection

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: ( A ) Images from representative time lapse movies of chromosomes (marked with H2B-mScarlet) and meiotic spindles (marked with MAP4 microtubule-binding domain (mNeonGreen-MAP4-MTBD)) in wild-type and REC8-FKBP12 F36V -mClover3 oocytes. ( B ) Representative metaphase II chromosome spreads with centromeres labeled by ACA staining in wild-type and REC8-FKBP12 F36V -mClover3 oocytes. ( C ) The percentage of aneuploid eggs matured in vitro from wild-type or REC8-FKBP12 F36V -mClover3 oocytes. ( D ) Representative images of metaphase II-arrested eggs matured in vitro from REC8-FKBP12 F36V -mClover3 oocytes treated with DMSO or dTAG-13 for 1 h and stained with ACA and Hoechst. ( E ) Quantification of the percentage of eggs with prematurely separated sister chromatids following 1-h dTAG-13 treatment during metaphase I followed by washout and progression into meiosis II. ( F ) Representative images of embryos at various stages of preimplantation development following fertilization of DMSO- or dTAG-13-treated REC8-FKBP12 F36V -mClover3 eggs. ( G ) Quantification of developmental progression from the zygote to blastocyst stage in embryos generated by fertilization of DMSO- or dTAG-13-treated REC8-FKBP12 F36V -mClover3 eggs. Data are from three independent experiments. Statistical significance was assessed using Student’s t-test. The number of analyzed cells or embryos is indicated in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Binding Assay, Labeling, Staining, In Vitro, Generated

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: a , Representative confocal images of REC8 (GFP antibody) and chromosomes (Hoechst) in metaphase I-stage REC8−FKBP12 F36V −mClover3 oocytes treated with DMSO (0 minutes) or dTAG-13 for 30 minutes, 60 minutes or 90 minutes. b , Quantification of normalized REC8 fluorescence intensity in metaphase I oocytes treated with dTAG-13 for the indicated durations, fixed immediately after treatment. Fluorescence was measured on chromosomes and normalized to DMSO control. c , Representative confocal images of metaphase II chromosomes (Hoechst) and centromeres (ACA) in oocytes treated with dTAG-13 for the indicated durations during metaphase I, followed by washout and progression into meiosis II. d , Quantification of the number of prematurely separated sister chromatids per metaphase II egg after the treatment and washout scheme in c . e , Integrated analysis of the data from b and d showing the relationship between REC8 fluorescence intensity and the number of prematurely separated sister chromatids per egg. REC8 levels were measured in metaphase I oocytes immediately after treatment, and corresponding chromatid separation was quantified in metaphase II eggs after washout and meiotic progression. Chromatid separation remained minimal until REC8 levels dropped below approximately 50% of control, beyond which separation increased sharply, supporting a threshold-based model of cohesion loss. Data are from three independent experiments. Statistical comparisons were performed using Student’s t -test ( b , d ). The number of analyzed oocytes is indicated in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Fluorescence, Control

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: ( A ) Representative confocal images of metaphase II-arrested REC8-FKBP12 F36V -mClover3 eggs treated with DMSO or dTAG-13 and immunostained with ACA (centromere marker) and HEC1 (kinetochore protein). Insets show magnified views of kinetochores. ( B ) Quantification of the proportion of oocytes exhibiting fragmented kinetochores after treatment. No significant increase in kinetochore fragmentation was observed in dTAG-13–treated REC8-FKBP12 F36V -mClover3 eggs compared to controls. ( C ) Representative images of γH2AX immunostaining in REC8-FKBP12 F36V -mClover3 metaphase II eggs treated with DMSO or dTAG-13, showing comparable levels of DNA damage marker signal. ( D ) Quantification of normalized γH2AX fluorescence intensity. No significant increase in γH2AX signal was detected in dTAG-13-treated REC8-FKBP12 F36V -mClover3 eggs. ( E ) Positive control: Representative γH2AX staining of wild-type metaphase II eggs treated with etoposide, a topoisomerase II inhibitor known to induce DNA damage, confirming assay sensitivity. Data are from three independent experiments. Statistical comparisons were performed using Student’s t-test. The number of analyzed oocytes is indicated in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Marker, Immunostaining, Fluorescence, Positive Control, Staining

Journal: Nature Aging

Article Title: A versatile cohesion manipulation system probes female reproductive age-related egg aneuploidy

doi: 10.1038/s43587-025-00997-w

Figure Lengend Snippet: a , Images from representative time-lapse movies of sister chromatids (H2B−mScarlet) in DMSO-treated and IgG TRIM-Away, DMSO-treated and CENP-A TRIM-Away, dTAG-13-treated and IgG TRIM-Away or dTAG-13-treated and CENP-A TRIM-Away eggs from REC8−FKBP12 F36V −mClover3 mice. t = 0 minutes denotes the start of live imaging experiment. b , Representative maximum intensity projected immunofluorescence images of CENP-A and chromosomes in metaphase II chromosomal spreads of DMSO-treated and IgG TRIM-Away, DMSO-treated and CENP-A TRIM-Away, dTAG-13-treated and IgG TRIM-Away or dTAG-13-treated and CENP-A TRIM-Away eggs from REC8−FKBP12 F36V −mClover3 mice. Boxes mark regions of interest that are 4.8× magnified. c , Quantification of CENP-A fluorescence intensities in metaphase II chromosomal spreads of DMSO-treated and IgG TRIM-Away (295 centromeres), DMSO-treated and CENP-A TRIM-Away (535 centromeres), dTAG-13-treated and IgG TRIM-Away (347 centromeres) and dTAG-13-treated and CENP-A TRIM-Away (465 centromeres) eggs from REC8−FKBP12 F36V −mClover3 mice. d , Measurements of chromatid scattering volumes from time-lapse movies of DMSO-treated and IgG TRIM-Away, DMSO-treated and CENP-A TRIM-Away, dTAG-13-treated and IgG TRIM-Away or dTAG-13-treated and CENP-A TRIM-Away eggs from REC8−FKBP12 F36V −mClover3 mice. Lines represent mean values, and error bars indicate s.e.m. Data are from three independent experiments. Statistical significance was assessed using two-way ANOVA ( c , d ). The number of analyzed oocytes is provided in brackets and italics.

Article Snippet: Primary antibodies anti-GFP (1:100 dilution; Roche, 11814460001), anti-phospho-histone H2A.X (Ser139) (1:250 dilution; Merck, 05-636) and human anti-centromere antibody (1:1,000 dilution; Antibodies Incorporated, 15-234) were incubated overnight at 4 °C.

Techniques: Imaging, Immunofluorescence, Fluorescence

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Survival profile of NSG mice. Mice were inoculated intraperitoneally with either B. abortus S19 or B. abortus S19ΔvjbR at various doses (1 × 104, 1 × 105, and 1 × 106 CFU/mouse) or with PBS. Mice inoculated with PBS or the B. abortus S19ΔvjbR vaccine candidate survived (100%) longer than mice inoculated with B. abortus S19.

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques:

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Temperature profiles of NSG mice inoculated with Brucella abortus. Mice were implanted with transponders, and body temperature was monitored every day over a period of 13 weeks following inoculation (i.p.) with B. abortus S19, the B. abortus S19ΔvjbR vaccine candidate, or PBS, as indicated. No significant changes in body temperature were observed (P > 0.05) over time in mice inoculated with B. abortus S19ΔvjbR or PBS. However, mice inoculated with different doses of B. abortus S19 demonstrated a gradual reduction in body temperature (hypothermia) that reached a significant level from week 7 onwards (***, P < 0.001). Doses are indicated such that S19-104, for example, indicates 1 × 104 CFU/mouse of B. abortus S19.

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques:

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Body weight profiles of NSG mice inoculated with attenuated Brucella abortus. Body weight was monitored over a period of 13 weeks postinoculation with 1 × 104, 1 × 105, and 1 × 106 CFU/mouse of either B. abortus S19 or the B. abortus S19ΔvjbR vaccine candidate or with PBS, as indicated. Mice inoculated with 1 × 105 and 1 × 106 CFU/mouse of B. abortus S19 demonstrated significant weight loss by 13 weeks. *, P < 0.05.

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques:

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Organ weight and bacterial colonization of NSG mice inoculated with Brucella. Mice inoculated (i.p.) with B. abortus S19 showed a significant increase in spleen weight compared with mice inoculated with the B. abortus S19ΔvjbR vaccine candidate (A), while there was no significant difference in liver weights in mice inoculated with Brucella or PBS (B). Mice inoculated with B. abortus S19ΔvjbR had significantly reduced bacterial burdens in spleen (C), liver (D), and lung (E) compared to those in the B. abortus S19-inoculated group. *, P < 0.05; ns, not significant.

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques:

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Gross image and tail histopathological image from NSG mice inoculated with PBS, B. abortus S19ΔvjbR (1 × 106 CFU/mouse), and B. abortus S19 (1 × 106 CFU/mouse), as indicated. (A) Representative image of NSG mouse inoculated with B. abortus S19 demonstrates inflammation in tail vertebrae. The magnified tail region with or without artificial coloring clearly demonstrates the regions of inflammation or bone damage (boxed selections). (B) Representative images of bone histopathology at low and high magnifications. Magnified images at right correspond with the boxed sections in the left panel. Top and middle panels depict intact bone mass and cartilage with normal appearance. Infection with B. abortus S19 induced severe bone resorption and destruction of the intervertebral discs which were replaced by fibrous connective tissue (bottom panel). Magnified images in the bottom right panel show neutrophil and macrophage infiltration (arrows) and activation of osteoclasts (arrowhead).

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques: Histopathology, Infection, Activation Assay

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Representative images of histopathology of spleen. (A) Representative bright-field image of spleen from immunocompetent mice (top) and from NSG mice inoculated with PBS, B. abortus S19ΔvjbR (1 × 106 CFU/mouse), and B. abortus S19 (1 × 106 CFU/mouse), as indicated. Images at right are magnifications of the boxed sections in the left panels. Mice infected with B. abortus S19 exhibited multifocal neutrophilic and histiocytic splenitis in contrast to mild neutrophilic and histiocytic splenitis in B. abortus S19ΔvjbR-inoculated mice. (B) Immunohistochemical localization of Brucella in mouse spleen (boxed).

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques: Histopathology, Infection, Immunohistochemical staining

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Representative images of histopathology of liver. (A) Representative bright-field image of liver from NSG mice inoculated with PBS, B. abortus S19ΔvjbR (1 × 106 CFU/mouse), and B. abortus S19 (1 × 106 CFU/mouse), as indicated. Images at right are magnifications of the boxed sections in the left panel. Animals infected with B. abortus S19 exhibited neutrophilic and histiocytic infiltration. None of the NSG mice inoculated with B. abortus S19ΔvjbR or PBS showed remarkable lesions. (B) Immunohistochemical localization of Brucella in mouse liver (arrows).

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques: Histopathology, Infection, Immunohistochemical staining

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Immunohistochemical, immunofluorescence, and fluorescence in situ hybridization (FISH) analysis of Brucella in paraffin sections of tail vertebrae. (A) Representative immunohistochemical images of tail vertebrae of NSG mice inoculated with PBS, B. abortus S19ΔvjbR (1 × 106 CFU/mouse) and B. abortus S19 (1 × 106 CFU/mouse). B. abortus S19-inoculated mice exhibited widely distributed Brucella antigens (brown) in the osteoarticular lesions, with the highest concentration in the osteoclasts (arrowhead). Only one mouse inoculated with 1 × 106 CFU/mouse of B. abortus S19ΔvjbR exhibited positive staining for Brucella (arrows). (B) Representative immunofluorescence images of tail vertebrae of NSG mice inoculated with PBS, B. abortus S19ΔvjbR (1 × 106 CFU/mouse), and B. abortus S19 (1 × 106 CFU/mouse). B. abortus S19-inoculated mice exhibited strong positive signal for Brucella antigens (green) that widely spread in the area of inflammation and osteoclasts (arrowheads), whereas NSG mice inoculated with B. abortus S19ΔvjbR exhibited a faint signal of Brucella antigens in few places (arrows). (C) Representative FISH image reveals hybridization of Brucella-specific Bru-996 Alexa Fluor-labeled DNA probe of the 16S rRNA gene with B. abortus S19 (red) in tail vertebral sections. Images at right are magnifications of the boxed sections in the left panel.

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques: Immunohistochemical staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Concentration Assay, Staining, Hybridization, Labeling

Journal: Infection and Immunity

Article Title: The NOD- scid IL2rγ null Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety

doi: 10.1128/IAI.00901-18

Figure Lengend Snippet: Brucella colonization in osteoclasts. (A) Representative immunofluorescence image of tail vertebrae of B. abortus S19-inoculated mice (1 × 106 CFU/mouse) demonstrated colocalization (yellow) of Brucella antigen (green) with the osteoclast marker cathepsin (red signal). Images at right are magnifications of the boxed section in the left panel. (B) Representative confocal immunofluorescence images showing a large number of B. abortus S19 bacteria (green) inside osteoclasts. The fluorescence intensity plot profile in the right panel corresponds with the images in the left panel. (C) Z sectioning shows Brucella localization in different depths of the osteoclast. AU, arbitrary units. (D) Quantitative image analysis shows a significant decrease (***, P < 0.001) in Brucella colonization in the B. abortus S19ΔvjbR-inoculated group compared with that in the B. abortus S19-inoculated group, as reflected in total pixel intensity.

Article Snippet: Blocked sections were incubated with polyclonal rabbit anti- Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature.

Techniques: Immunofluorescence, Marker, Fluorescence